WO1991014359A1

WO1991014359A1 - Process for producing tuber

Info

Publication number: WO1991014359A1
Application number: PCT/JP1991/000382
Authority: WO
Inventors: Noboru Onishi; Kunihiro Hayashida; Kanji Mamiya
Original assignee: Kirin Beer Kabushiki Kaisha
Priority date: 1990-03-23
Filing date: 1991-03-25
Publication date: 1991-10-03
Also published as: ES2190444T3; DE69133173T2; EP0691073A3; DE69133173D1; EP0476141A4; EP0476141B1; DE69116044T2; DE69116044D1; EP0691073B1; EP0476141A1; BR9105109A; US5862626A; EP0691073A2; ES2083571T3

Abstract

A process for producing tubers of a solanum readily in a large amount, wherein the stolon and foliage which have grown in a liquid phase under application of carbon dioxide in the light period of the foliage growth stage are exposed to a gas phase in the tuber formation stage to form tubers in a high yield, and the culture in the tuber formation stage is conducted while aerating the gas phase of a culture tank to form a large amount of tubers not only near the boundary of the culture medium but also in the gas phase apart from the medium. As a result, the efficiency of tuber formation and the quality of the tuber are improved.

Description

Details

How to produce tubers

[Industrial applications]

The present invention relates to a method for producing tubers of Solanum plants.

[Conventional technology]

At present, world potato production is mainly based on its vegetative reproduction. In other words, when tubers, so-called potatoes, which are vegetative breeding organs, are planted, as the plants grow, from the nodes (axillary buds) in the underground to the stron (a kind of lateral technique, the lateral technique on the ground is not On the other hand, the stalks have the same morphology as the main stem, whereas the stront extends obliquely in the ground horizontally or diagonally downward and has no clear leaves. It enlarges and tubers are obtained again. These are used as next-generation seed potatoes, or for food and processing. Normally, about 10 tubers are reproduced from one plant.

In such vegetatively propagated potatoes, various viral diseases, which are mainly borne by aphids, have become a very serious problem worldwide. In other words, seed-breeding crops such as rice, wheat, and corn, even if the plant is infected with a virus disease, most of the virus is not found in the seed formed on the plant by fertilization. Does not shift, so the impact on the next generation is not so large On the other hand, potatoes propagate mainly in tubers without fertilization as described above, so once infected with the virus, the virus propagates in the body for generations, causing serious damage to the harvest. It is almost impossible to remove such a potato-infected virus from the plant under normal natural conditions, so it is now possible to infect as much virus as possible to reduce the harm of the virus disease. Uncultivated cultivation systems are used worldwide. The basis is the supply of virus-free seed potatoes. In other words, no matter what kind of virus control system is used in potato cultivation, if the seed potatoes to be planted are contaminated, the effort is meaningless for the above-mentioned reasons.

The supply of “Virus Free” seed potatoes is generally carried out in the following manner, although there are some differences in each region.

(1) Production of virus-free individuals by growing point culture, (2) adaptation of dirt-free individuals, production of small tubers by potting, (3) cultivation of these small tubers in a greenhouse, harvesting of tubers, (4) Cultivation and reharvesting of the obtained tubers in the field. (5) Continue cultivation in the field according to the required amount (3-5 times). As described above, the world's first virus-free seed potato production is currently being carried out, but since this method has the danger of re-infecting the virus in the field, complete cultivation requires advanced cultivation techniques and techniques. Quarantine systems are indispensable and simpler and safer methods It has been demanded.

As one of the methods, use of tubers produced under aseptic conditions by tissue culture has been studied in various fields. That is, the virus-free individuals obtained by the growth point culture are not exposed to a greenhouse, a field, or the like, and tubers are produced as they are in vitro (iv vitro). It is cultivated twice in the field, and the resulting potatoes are used as seed potatoes. Tubers formed under these aseptic conditions are significantly smaller (typically 1 g or less in diameter) than conventional tubers (tuber, tuber, 50-300 g) by conventional methods. Therefore, it is also called "micro tuba".

These tubers have the following advantages. (1) Tubers are completely sterile, including virulents, because they are made from asphyllous plants under sterile conditions. (2) Since tubers are produced under tissue culture conditions, they can be produced throughout the year regardless of the quantity and quality of natural conditions. (3) Tubers can become seed potatoes directly or have the potential to produce seed potatoes by cultivation once or twice in the field, so there is no risk of re-infection with viruses or other diseases. Or it is smaller than the conventional method.

(4) Since tubers are small in size, they can be easily transported in large numbers.

Tubers produced under aseptic conditions that have these advantages are generally divided into two major steps as follows: (Estrada et al., 1986. P1 ant Cell Tiss. Org. Cult. 7: 3-10, Wang PJ and CY Hu. 1982. Am. Potato J. 59: 33-37, Rosell et al. 1987. Potato Research 30: 111-116 ^ Hussey G, and NJ Stacey. 1984. Ann. Bot. 53: 565-578).

Foliage growth step: This step is to grow the potato foliage itself in order to increase the axillary buds necessary for tuber formation later, that is, the buds contained in each node. This process involves adding 1 to 3% sucrose as a carbon source to a basal medium usually used for plant tissue culture, and sometimes a low concentration of plant hormones, and adding a constant amount of agar to agar or liquid. It is performed under photoperiod of light. A plant piece with at least one bud planted under this condition grows in a form similar to that of the aerial part under natural conditions, with a similar reduced size. Although rooting is also involved, tuber formation does not usually occur under these conditions. The culture temperature is 20-25. The culture period is usually 3 to 5 weeks.

Tuber-forming step: The above-mentioned base containing sucrose (5 to 10%) higher than that in the foliage-growing step, and optionally containing plant hormones such as cytokinin or dwarfants Transfer to medium. When the foliage growth process is performed in liquid, the medium is replaced with the same component. Temperature is 15 ~ 25 '(: generally in dark condition, sometimes low light or short of 100 ~ 5001ux Day conditions are used. The culture period is usually 4 to 8 weeks. By these operations, tubers are formed directly on the axillary buds contained in the foliage or mainly on the stems extended from them.

However, the conventional method has low tuber induction efficiency, and the method itself is not suitable for producing a large amount of tubers. As a result, the cost of producing tubers is high, and it is not a practical technique. These conventional techniques have only been used for preservation and distribution of gene sources.

In recent years, attempts have been made to improve the disadvantages (Japanese Patent Application No. 63-500104 (International Publication No. W088 / 04136), Akita M. and S. Takayama. 1988. Acta Horticu lt. 230: 55- 61). In the improved method, most of the tubers are formed only near the interface between the culture medium and the gas phase, so to efficiently induce tubers in long elongated plants, the medium surface must be gradually grown in the tuber formation process. We need to lower it. For this purpose, the method used here is to grow the plant, add a tuber-forming medium so that most of it is submerged, and artificially transform the medium when tubers begin to form near the boundary interface. After the tuber has formed near the surface of the medium, the operation is repeated, or a large amount of air (0.8 to 2.2 vvm) is passed through the liquid phase to force the medium. In this method, the medium is gradually evaporated and the surface of the medium is gradually lowered.

In these methods, the majority of the media used is mainly used only for the induction of tubers, and is less often used for hypertrophy. Absent. In other words, the added nutrients and carbon sources are not fully utilized, and it is difficult to further reduce production costs. In particular, the method of gradually and artificially removing the culture medium requires many operations, and increases the risk of contamination. Most of the axillary buds, which mainly form tubers, are formed in the gas phase during the foliage growth process, and the foliage formed in the medium is hardly used. In addition, in our study, tubers produced near the boundary of the culture medium were not suitable for storage and field cultivation, depending on the cultivar.

[Problems to be solved by the invention]

An object of the present invention is to provide an improved method for producing tubers which has solved the above-mentioned problems of the prior art. More specifically, it is an object of the present invention to provide a method for easily producing a large amount of tubers excellent in storability and cultivation.

[Means for solving the problem]

The present inventors have conducted researches to achieve the above object. As a result, surprisingly, the application of carbon dioxide gas during the light stage of the foliage growth process was surprising. Strongly inducing and proliferating stron under feeding conditions; (2) Inducing and proliferating thicker strons more efficiently under conventional stron inducing conditions; (3) Long day Inducing and multiplying the stolon more vigorously under low temperature conditions in the dark period. (4) Under the application of carbon dioxide gas By exposing the strons and foliage extending to the liquid phase to the gaseous phase during the tuber formation process, the exposed stron and stalk are synchronized in a short period of time with a remarkably high rate of tubers. Found to be molded. Also, surprisingly, in the tuber formation process, culturing under the conditions of direct aeration into the gas phase of the culture tank, regardless of the presence or absence of the stron and the exposure of the foliage to the gas phase, (5) ) We found that a large number of tubers were formed not only near the boundary of the culture medium but also in the gas phase away from the culture medium surface, and as a result, the efficiency and quality of tuber formation were improved. Furthermore, (6) it has been found that the combination of the above (4) and (5) enables excellent tuber production in terms of quantity and quality, and the present invention has been completed based on the above findings. Was.

So far, there have been several reports on culture methods using carbon dioxide gas (JP-A-285117, JP-A 1-304826, JP-A 2-16921, Lakso et al., 1986. J. Amer. Soc. Hort. Sci. Ill: 634-638, Kozai T. and Y. Iwanami. 1988. J. Japan, Soc. Hort. Sci. 57: 279-288) ₀ However, the purpose of any of these methods is By shifting the culture of conventional heterotrophic growth to light-independent growth, the growth of plants can be accelerated, rooting can be promoted, and the survival rate during acclimation can be improved. Yes, carbon dioxide is also used to optimize photosynthesis in a culture environment. That is, for that purpose, the cultured plant has the same morphology and properties as the plant under natural conditions. It has been reported that the application of carbon dioxide gas induces new forms of plants that cannot be recognized under natural conditions and uses them. I was not even heard.

Although potatoes have been reported to be cultured under the application of carbon dioxide gas (Kozai et al. 1988. Acta Horticult. 230: 12 1-127, Koza et al. There is no example used for tuber formation. The purpose of these reports is the same as in the previous example, and it is conceivable to grow cultured plants in the same form as the above-ground part under natural conditions, which can be easily adapted to natural conditions by imparting light-independent growth. ing. That is, in this method, stron induction, proliferation, and tuber formation from them have not been observed, and the possibility thereof has not been suggested. The concentration of carbon dioxide used in these reports is set for the purpose of optimizing photosynthesis of plants under certain culture conditions, and the concentration is mainly 100 ppm (0.1%). The following relatively low values are illustrated.

Carbon dioxide treatment of the root system (Arteca et al., 1979. Science 205: 1279-1280) and the normal tubers themselves (DR Paterson 1975. J. Amer. Soc. Hort. Sci. There are reports that have a positive effect on the growth of the head, the length of the stron, the development of the stolon, the number of tubers formed per stron, and the tuber yield. Even in this case, the plant shows a form in which the growth part of foliage and the formation part of the tuber are clearly distinguished, which can be said to belong to the same range as the form of potato under natural conditions. Also, in these reports, high concentrations of carbon dioxide (the former -45, the latter-80%) were applied to specific organs (the former-the root system, the latter-the tubers) for a short time (both 12 hours).

0.

On the other hand, under culture conditions, strons have been observed to be mainly formed under short-day conditions, but they are generally thin, have low induction efficiency, and have poor tuber-forming ability. Since it is not high, it has not been actively used for tuber production. In addition, under long-term conditions, which are generally said to promote the growth of potato corn under natural conditions, under culture conditions, stron elongation and tuber formation have been rarely observed (see above, Hussey G. and Ν · J. Stacey), but there is no example of efficiently inducing and growing strons and using them for tuber production. In addition, there are no culture examples of tuber production that combine low-temperature darkness with long-day conditions.

However, the present inventors have found that an effective plant morphology for tuber production can be induced by applying carbon dioxide gas during the light period. In other words, under natural conditions, it only elongates mainly to the root system in the soil, and during culturing under the same conditions (mainly long-day conditions) without the application of carbon dioxide, it hardly occurs. Unformed strons, and even under some conditions (mainly short-day conditions), are more efficient with thinner and fewer strons, thicker and thicker, from the top to the bottom of the plant. It has been vigorously differentiated and developed. As a result, good conditions can lead to dense and distinctive forms of strons (Figures 3 and 4).

Furthermore, it was also found that the axillary buds contained in the stron and stem obtained by the method of the present invention had higher tuber-forming ability than those obtained by the conventional method. In particular, the most effective (short-time, synchronized and high-efficiency) is obtained from the axillary buds contained in the strons and stems that have been grown under the conditions that elongate into the liquid phase during carbon dioxide application and are exposed to the gas phase during tuber formation. Tuber was formed in the gas phase regardless of the distance from the medium surface. Under such conditions, tubers are mainly formed by direct enlargement of the tips of the strons, but tubers can also form from the strons and the axillary buds of the stems, either directly or through new stalks. Many were formed. As a result, in good cases, a large part of the plant from the top to the bottom is covered with tubers, that is, the formation of foliage and tubers is clearly distinguished in potato growth under natural conditions. It showed a very characteristic form in which parts were mixed (Fig. 5). This phenomenon is due to the fact that most tuber formation in the conventional culture tank occurs in the axillary buds, which existed in the gas phase during the foliage growth process, and near the medium boundary in the tuber formation process. It is very different.

As described above, the induction and proliferation of stron in the present invention, and the induction of dense morphology of stron and tuber are performed in the plant culture method including carbon dioxide application and the tuber formation method described above. No tuberculosis was reported, nor was it possible to predict from them, and the tuber formation efficiency was greatly improved. In addition, in this method, the tuber induction medium, which generally uses a higher concentration of sucrose than the foliage growth medium, can be used in a smaller amount than the conventional method, and the tuber formation efficiency per unit sugar amount is significantly improved. did.

On the other hand, the conventional method, in which ordinary air that is not enriched with carbon dioxide, is directly ventilated to the gas phase in the culture environment, also aims to improve the growth rate of cultured plants and the survival rate after acclimation by promoting photosynthesis. No change in plant morphology is considered. In addition, there is no example in which a method of directly aerating the gas phase is used in the tuber forming step under culture conditions.

However, direct aeration into the gas phase of the culture tank during tuber formation according to the present invention was unexpected in that it led to characteristic plant morphology different from that under natural conditions and greatly improved tuber formation efficiency. . In other words, when the gas in the culture tank is appropriately ventilated, regardless of the amount of the medium added, and without significantly lowering the medium surface, the portion between the top of the plant in the gas phase and the area in contact with the medium can be used. Many tubers were formed. At that time, tubers grow during the pre-plant growth. In some cases, the stems are formed directly on the axillary buds of the elongated stems, or in those cases, new stems or stodones from the axillary buds elongate during this process and are formed on them. In addition, in the preferred method of direct aeration into the gas phase, tubers are formed under relatively dry conditions due to reduced humidity of the tuber-growth area. High quality tubers with less damage to the epidermis can be obtained.

This type of tuber formation can achieve similar tuber formation under culture conditions using a conventional culture tank without artificially lowering the boundary surface of the culture medium. No, it's a new phenomenon.

This method at the time of tuber formation is effective regardless of the state of the plant used, that is, the culture method of the foliage growth process (Fig. 6), but it is combined with the tuber production method using carbon dioxide as described above. The best results are obtained (Fig. 5).

That is, the method for producing tubers of the present invention is a method for producing tubers of a Solanum genus plant having tuber-forming ability, as described in the following claims:

"1. A method for producing a tuber of a Solanum plant having a tuber-forming ability comprising a step including a foliage growing step and a tuber forming step in a culture vessel,

(1) In at least a part of the foliage growing step, a plant piece having at least one bud is Cultivated in a liquid medium containing a carbon source and inorganic salts, with carbon dioxide applied to at least part of the light period, to induce and proliferate foliage and stront,

(2) In the tuber formation step, at least a part of the stron formed in the liquid medium in the foliage growth step is exposed to a gaseous phase, and cultured in a liquid medium containing at least a carbon source. Tuber production method characterized by the following:

2. The method for producing tubers according to claim 1, wherein in the tuber forming step, the relative humidity of the gas phase is reduced by aerating the gas phase to culture the tuber. 2. The method for producing tubers according to claim 1, wherein the applied concentration of carbon dioxide is 0.1 to 30% (v / v). The method of applying carbon dioxide gas is characterized in that 0.1 to 30% (v / V) of carbon dioxide gas is aerated at a rate of 0.001 to 1 volume per minute with respect to the volume in the container. The method for producing tubers according to claim 1.

In a method for producing tubers of a Solanum genus plant having tuber-forming ability comprising a step including a foliage growing step and a tuber forming step in a culture vessel, the tuber forming step includes aerating the gas phase in the tuber forming step. A method for producing tubers, which comprises culturing while reducing the relative humidity of the gas phase. J

It is characterized by including. Hereinafter, for convenience of explanation, the invention described in claim 1 is referred to as a first invention, and the invention described in claim 5 is referred to as a second invention.

Hereinafter, the present invention will be described in detail, but the stone used here will be defined.

Stron: The following three types of stems are called “strons” here (Figs. 3 and 4).

(1) It has a morphology similar to the stron of a plant that grows under natural conditions. (Long internodes compared to the side technique on the ground. It has a hook-junction at the tip, and leafy green There is no body and it is almost white. The leaves of each node are suppressed to the extent of traces.) It shows a clear oblique orientation (property that extends horizontally or downward).

(2) Most forms are the same as those of (1), but have slightly developed leaves with chloroplasts, and sometimes have a partial red color. The direction of extension shows obliqueness.

(3) It has the same form as the stron of (2), but extends upward, and no further leaf development is observed on the medium or even when it comes out on the medium, or its development But very slow.

Plants of interest in the present invention include all species of tuber-forming ability of the genus Solanum. Specific examples of such plants are, for example, potato cultivars (So lanum t uberosum), S. demi ssum, S. acaule, S. stol on iferum, S. phure ja and the like. Of these Is a representative of potato cultivars.

In the case of a culture tank, the container used in the present invention may be any container that can ventilate air or air enriched with carbon dioxide, but also ventilates the liquid phase and gas phase of the culture medium. It is desirable that it is possible. When a culture tank is not used, any air-permeable container or cap for ordinary plant tissue culture may be used. Ordinarily, an Erlenmeyer flask covered with a lid with good air permeability, a cylindrical culture bottle or a culture container made of polycarbonate is used.The first invention is to apply carbon dioxide gas during the light period to grow plants, And a stron inducing and multiplying, a foliage growth step (step (A)) and a tuber formation step (step (B)) for forming tubers on the axillary buds contained in the stron and stem of the plant. Therefore, they are described separately below. In addition, the second invention will be described in the following step (II). Further, the inventions described in claims 2, 3 and 4 other than the first invention and the second invention will be described in the following steps (I) and (II).

(Α)

The plant material used in this process can be of any origin, provided that it is maintained aseptically, but is usually derived from an individual growing in the field or from a growth point culture of buds sprouted from harvested tubers. The body is used to protect against viruses and other diseases It is desirable to be completely free. In this step, roots are not necessarily required as long as the plant pieces have at least one bud, but it is preferable to use plantlets having several buds and roots as planting materials. good. The number of seeds used is 1 to 50 per liter of medium. The plant or plant piece may not have a stron formed thereon, but preferably has a stron formed therein. This is because when using plant pieces that do not have a mouth as a material, it takes a certain amount of time to “induce” strons that did not exist before, and then the strons and foliage While vigorous stront "proliferation" occurs, in the case of a material containing a stron, the "induction" of the stron has already taken place, so the "proliferation" of the stron immediately occurs after placement. This begins because the culture time can be used efficiently. Further, it is needless to say that a stron or a part of a stron having at least one bud may be used as a planting material in this step. By this step, foliage and stron are induced and propagated, and their tuber-forming ability is further enhanced.

The medium used in this step may be a basic medium usually used for plant tissue culture supplemented with 1 to 3% of sucrose as a carbon source. The basic medium is Murashige &Skoog's medium (hereinafter referred to as M S medium), Linsmeier & Skoog medium, White medium, Schenk & Hill debrandt medium, etc. Usually, an MS medium is used. The composition of these conventional media is described, for example, in Harada and Komine, "Plant Cell Tissue Culture", pp. 390-391, RIKEN, 1984. Examples of carbon sources other than sucrose include glucose, maltose, molasses and the like.

These basal media contain plant growth regulators such as 1-naphthalinacetic acid (NAA), indole-3-acetic acid (IAA), and 2,4-dichlorophenoxyacetic acid (2,4-D ), Lee emissions doll one 3 - butyric acid (I BA :), Jibere-phosphate (GA _3), 6 - base Nji Ruadenin (BA), a plant hormone Ya Sa Ikoseru such force Inechin (CCC), such as Anshimi Doll A dwarfing agent may be added in the range of 0.001 to 10 ppm. In particular, GA ₃ is often effective for elongation of the stron. The amount of medium used is not particularly limited, but (1) strons are more easily and more induced in the medium than outside the medium, and (2) strons grow vigorously in the solution. (3) The stems other than the stront vigorously elongate and branch in the liquid, (4) The stem contains many axillary buds, (5) ) Tubers are more efficiently formed from the axillary buds contained in the sponge and stems formed in the liquid under the conditions described below. Is desirable. Medium pH should be 4-8, preferably 5-7

In addition to the basic culture conditions described above, photoperiod, temperature, and the direction of light irradiation are related to the induction and proliferation of strons.

1. Long day conditions

Although it depends on the photoperiod sensitivity of the cultivar, strons are generally less likely to be induced under the long-day conditions normally used for potato tissue culture, i.e., at a photoperiod of 14 hours or more and at 20 to 25 ° C. . Also, when induced, the stron elongates as above-ground or foliate stalks, and hardly elongates as a stron. Vigorous stron induction and multiplication by long-day application of carbon dioxide is remarkable under the condition that (1) continuous day length or (2) the temperature in period (1) is 15 ° C or less. Is recognized.

In the case of (1), the culture temperature is 15 to 30, preferably 17 to 22 ° C, and in the case of (2), the light period is 15 to 30, and the dark period is 5 to; 15 ° C, preferably each A temperature change condition of 17 to 25 ^e C and 8 to 12 is preferable. The illuminance is preferably 1000 to 20,0001 ux for both (1) and (2), and more preferably 3000 to 80,001 ux. The light period of (2) is preferably 14 to 20 hours, and more preferably 15 to 18 hours. Since the strons induced by this method include those that extend in the direction of light irradiation, illumination from the lateral direction or from below is preferred to increase the number of storons present in the liquid. 2. For short-day conditions

Under short-day culture conditions, usually less than 12 hours, many varieties induce strons and elongate them as strons. However, straws obtained without carbon dioxide application are generally thin, small in number, and low in tuber formation efficiency. When combined with carbon dioxide treatment under short-day conditions, the strons become significantly thicker and more numerous, and the tuber formation rate is significantly improved thereafter. The daylength is 6-12 hours, preferably 8-10 hours. Since the growth of the plant itself tends to be delayed under short-day conditions, a plant cultured for a certain period under long-day conditions may be used. Temperature is 5-30 ° C, preferably 17-25. It is more effective to lower the temperature during the dark period to 5-15 ° C. Illuminance is 1000 to 20,0001 ux, preferably 3000 to 80,001 ux. Stones guided in this way are likely to show geotropism or skew. The light may be emitted in any direction, but illumination from the side or from below is also effective.

The method of applying carbon dioxide is common for long day and short day conditions. In the case of a culture tank, use a method of culturing in a carbon dioxide-enriched atmosphere, while in the case of a small culture vessel, a condition in which air rich in carbon dioxide gas is passed. The concentration of carbon dioxide used for ventilation in the culture tank or in the atmosphere for culturing the permeable culture vessel is within the range of 0.1 to 30% (v / v), preferably 0.5 to 20% (v / v). / v). The amount of aeration to the culture tank is Use 0.0001 to 1 volume per minute per unit volume of the product, preferably 0.001 to 0.1 volume.

Under these conditions, a large number of axillary buds are efficiently induced and proliferated, which can be easily converted into tuber via the stron in the gas phase and liquid phase, especially in the latter, and in the subsequent tuber formation process via stron. You. If the concentration of carbon dioxide is lower than this, or if the aeration rate is low, the plant will show a normal growth form and a stron will not be formed. In other words, the buds extending from the axillary buds in the liquid do not become stront but reach normal foliage. The ability of the axillary buds contained in these stems to form tubers is low.

Conversely, if the carbon dioxide concentration is higher (over 30%), the plant will die. If the airflow is higher

(1 volume per minute or more per unit volume) is caused by excessive harm of carbon dioxide gas and a large amount of ventilation, evaporating the culture medium, over-humidification of the gas phase (in the case of ventilation to the liquid phase), Or, conversely, excessive drying (in the case of aeration into the gas phase) delays plant growth and elongation of the stron.

In the case of a culture tank, the ventilation of the coal gas-enriched air may be divided into the liquid phase only, the gas phase only, or the liquid phase and the gas phase. Desirably, a method in which carbon dioxide gas-enriched air is passed through the gas phase and a suitable amount of ordinary air, ie, about 0.001 to 0.05 vvm, is passed through the liquid phase.

The application of carbon dioxide is at least part of the foliage growth process In addition, it should be carried out at least one hour per day, preferably two hours or more per day. The application period is between 10 and 70 days, preferably between 20 and 40 days.

The size of the plant or plant piece to which carbon dioxide is applied is not particularly limited. That is, the application of carbon dioxide may be started immediately after the plant body or the plant piece is placed, or the carbon dioxide gas may be applied by the above-mentioned method after cultivation for a certain period after planting by a known method.

If there is a period when carbon dioxide is not applied, normal air may be ventilated under the same conditions. In the case of a culture tank, it is desirable to ventilate at least an appropriate amount, that is, 0.001-1.

(B) Process

As the tuber induction medium in this step, a medium containing at least a carbon source as an essential component and having the same or higher sugar concentration as in step (A) is used, but is not limited thereto. In the case of sucrose, the sugar concentration is 5 to 15%, preferably 6 to 10%. In some cases, plant hormones such as 6-benzyladenine (BA), potato-inetin, zeatin, abscisic acid (ABA), and dwarfants such as psychocell (CCC :) and ansimidol can be used in the range of 0.01 to 20 ppm. May be used in The medium used in step (A) is removed from these mediums, and at least the strains that have been induced, grown, or have enhanced tuber formation in the medium in step (A) are removed. A part is added so as to be exposed in the gas phase. That is, under the conditions of the present invention, the strons elongated in step (A) already have a high tuber-forming ability irrespective of the elongation position, but mainly the strons and stems elongated in the liquid phase part in this step. Exposure into the phase allows the formation of many strons and stems in the gas phase, since their location is very independent of their distance from the culture medium and from which tubers are formed in a short period of time, synchronously and very efficiently. It is better that the amount of culture medium is moderately smaller than that in (A) so that the medium is exposed. Usually, use a medium in an amount of 5 to 80%, preferably 10 to 40% of the medium in step (A).

Further, the medium used in step (A) may be reduced so as to satisfy the condition. Further, a necessary amount of a concentrated solution of a component that promotes tuber induction (sugar, the basic medium component, a plant hormone, etc.) may be added to the medium. Apart from the operation of the amount of the medium, a method may be used in which the stron is exposed in the gas phase. For example, the plant can be pulled up from the medium, the plant can be inverted, turned upside down, the entire culture tank can be turned down using a vertically long culture tank, or the shape can be changed using a bag for culture. The pH of the medium is 4-8, preferably 5-7.

Various conditions can be used for the light environment. In other words, low light conditions of 100-l OOOlux are within an appropriate day length range (4-24 hours), and short-day conditions of 4-10 hours are within an appropriate light range (100-8801 ux). Can be used in More preferably, good results are obtained when both are combined, or when only the portion where tubers are formed is shielded from light. The 喑 condition also produces good results. Especially under short-day conditions, many tubers are formed without increasing the sugar concentration in the medium.

The temperature condition is 5 to 30, preferably 15 to 25 ° C. When using short day conditions, the dark period temperature may be lower than the light period temperature.

In this step, when a container having air permeability is used, it is preferable to culture the cells in an air environment satisfying the above culture conditions. In the case of the culture tank, it is usually sufficient to ventilate the liquid phase in the culture tank (the amount of ventilation is, for example, 0.001 to 0.1 lvvm).

In the step (B), even better results can be obtained by combining the second invention. That is, in the above tuber formation step, it is preferable to lower the relative temperature of the gas phase by ventilating the gas phase. The optimal amount of aeration at that time depends on the absolute volume of the culture medium, the shape of the container, the amount of plants present in the gas phase, the amount of ventilation to the liquid phase, the number and shape of the vents to the gas phase, etc. However, each may be 0.02 to 1 volume per minute per unit volume, preferably 0.05 to 0.5 volume. Furthermore, when the relative humidity of the gas phase falls to 90% or less due to this ventilation, better qualitative and quantitative results are often obtained in tuber formation. Below this ventilation In tuberculosis, tubers are not sufficiently formed in the gas phase, and the plant will die due to excessive drying. When ventilating to the liquid phase, it is preferably 0.05 vvm or less. Under the above conditions, the medium does not evaporate significantly and does not decrease. Further, even if the aeration rate is such that the amount of the culture medium is reduced, when the gas phase is aerated, more tubers are formed than in the culture using only the liquid phase.

The second invention can be used for a plant produced by a conventional culture method, and has a remarkable effect.

In addition, in the present invention, instead of the forced aeration into the gas phase in the tuber formation step described above, other means capable of culturing at a relative concentration of 90% or less (for example, (1) ) Use of a culture tank with high ventilation capacity, (2) Use of a substance that suppresses evaporation of water from the medium surface, (3) Use of a culture tank in which water evaporated from the medium surface is difficult to transfer to the gas phase) May be used.

The culture period in this step is 10 to 50 days, preferably 20 to 40 days.

[Brief description of drawings]

Fig. 1 is a diagram (photograph) showing the state of foliage growth by the conventional method (the method of the control plot in Example 2), and Fig. 2 is a tuber by the conventional method (the method of the control plot in Example 2). Fig. 3 shows the state of formation (photo), and Fig. 3 shows the state of induction and proliferation of strons by carbon dioxide application in Example 6 (photo). FIG. 4 is a diagram showing the state of induction and growth of stron by carbon dioxide application in Example 6 (photograph taken from the bottom of the culture tank), and FIG. 5 is a diagram showing the state of carbon dioxide application in Example 4. Fig. 6 shows the state of tuber formation of the induced and proliferated stron and stem (photo) and Fig. 6 shows the state of tuber formation by aeration to the gas phase of the culture tank of Example 1 (photo). It is.

〔Example〕

Hereinafter, the present invention will be described in more detail by way of examples, but these examples do not limit the scope of the present invention at all.

Example 1

Influence of ventilation to gas phase in culture tank during tube formation on tuber formation position and efficiency

As a test material in this experiment, a sterile plant of a potato (So lanum t uberosum, cultivar: Norin 1) obtained from a growing point culture was used.

Three plantlets with 5 nodes and roots, containing 10% MS liquid medium (pH 5.8, hereinafter referred to as foliage growth medium) supplemented with 3% sucrose, 10 ^, diameter 27cm, height 40cm the total capacity of about 23 ^ were plated into a cylindrical culture vessel, 22 ^e C, illuminance 60 OO l ux, 16 hr photoperiod, in a liquid phase 30ml / min and the gas phase 500 ml / min of aeration conditions, 5 weeks stover Was grown. By this culture, the plants grew vigorously to a height of about 30 cm. next Replace the remaining medium with 10 ^ MS liquid medium containing 8% sucrose (pH 5.8, hereinafter referred to as tuber induction medium) 10 ^, and induce tuber induction to 22, dark place and (1) liquid phase only. Aeration of 1030 ml (control), (2) Aeration to the liquid phase 30 ml / min, aeration to the gas phase lOOOinlZ 3 weeks for 3 weeks, and compared the tuber formation position and its efficiency . Ventilation to the gas phase was performed from the top both during foliage growth and tuber induction, and the exhaust port was set at a height of about 30 cm. At the end of the culture, the medium surface was almost the same in both (1) and (2). The results were as shown in Table 1.

(Margins below this page)

Table 1

*… Distance from the culture surface at the end of culture

Tubers were formed directly on the axillary buds in the gaseous phase in the aerated section, and white stalks without chloroplasts grew vigorously from many axillary buds in the liquid and gaseous sections. However, many tubers were also formed at the nodes existing in the gas phase. In contrast, in the control plot, tubers were hardly formed on the axillary buds in the gas phase, and although similar white stems were seen, the number was small and many of the top buds died due to the high humidity, and the tubers Most were formed on the axillary buds at and just above the medium interface. As a result, the number of tubers and tuber weight in the gas-phase aerated area improved significantly more than twice as much as in the control, and the formation position was also significantly different from that in the control. There were many tubers even in the area of 10 cm or more, which was not recognized at all. Almost all of the tubers in the control plot showed remarkable skin hypertrophy and secondary growth, but the tubers in the gas-phase aerated plot were also significantly improved in that respect.

Example 2

Influence of ventilation to the gas phase inside the culture tank during tube formation on tuber formation efficiency and quality

A foliage growth medium obtained by dividing a sterile plant of Solanum tuberosum L. (variety: Russet Burbank) into plantlets having roots and five nodes, and changing the five of them to a sucrose concentration of 2%. They were placed in an 8 ^ culture tank (diameter 20 cm, height 24 cm) containing 3. 30mlZ min in liquid phase, gas phase The part was aerated at a rate of 100 ml / min, and the foliage was grown for 5 weeks at 22, illuminance of 6000 lux and 16 hours of photoperiod. Next, after replacing the medium with the tuber induction medium 3, the air volume in the gas phase was changed to 1 ^ / min, and tuber formation was performed at 22 places for 3 weeks at 22 places. As a control, a section was provided for cultivation under the same conditions except that aeration during tuber induction was performed only in the liquid phase with SOml Z. The results are shown in Table 2. Table 2

Tubers were formed mainly in the gas phase in the gas-phase aerated section through the same process as in Example 1, and formed unevenly near the boundary of the culture medium in the control section. The gas-phase aeration group exceeded the control group in both total tuber number and weight, but there was a remarkable difference particularly in the degree of damage to the epidermis of the tuber. In other words, most of the tubers in the control group had remarkable hypertrophy and damage to the epidermis due to overhumidity or contact with the medium, and secondary growth was large, whereas the degree in the gas-phase aeration group was slight. I stayed something. This difference was more pronounced under tuber storage conditions. More than half of the tubers in the control plots due to excessive water evaporation during storage at low temperatures. The tuber in the gas-phase aerated area showed little change in quality qualitatively.

As described above, the method of forming tubers in the gas phase has a remarkable improvement in the quality of tubers in varieties such as bursett bar banks, which have weak tubers (easily damage the epidermis) under humid conditions.

Example 3

Effect of relative humidity of gas phase in culture tank on tuber formation during tuber formation

After growing the foliage to a height of about 20 cm under the same conditions as in Example 1 except that a culture tank with a diameter of 20 cm, a height of 48 cm, an internal volume of about 16 ^, and a foliage growth medium of 3 were used, the tuber induction medium of 3 was used. The remaining medium was replaced with the remaining medium, and the position of the gas phase aeration was changed in culture in a dark place, the liquid phase aeration 30 mlZ minutes, and the gas phase aeration 1 ^ Z minutes, resulting in the relative humidity. The effect of the difference in the tuber formation on tuber formation was compared. In other words, there are two methods of venting the gas phase (1) near the surface of the culture medium (approximately 5 cm above) and (2) near the top of the culture tank. Also in the direction of the culture medium). The relative humidity (measured by the exhaust port installed near the top) and the degree of tuber formation at that time were investigated. The results are shown in Table 3.

(Margins below this page) Table 3

*… Distance from the culture surface at the end of culture

In the section (2) ventilated from the vicinity of the top of the culture tank, the white stems similar to those in Example 1 also vigorously elongated, most of them reached the top, and a large number of tubers formed near the same. In section (1), which was aerated near the surface of the culture medium, tubers formed at higher positions were rare. At that time, in section (1), fine water droplets were remarkably adhered to the upper part of the culture tank, and the relative humidity measured at the exhaust port was significantly lower in section (2) than in section (1). This result indicates that aeration into the gas phase with lower humidity in the space where tubers are formed is more effective.

Reference example

Examination of the productivity of tubers produced by the gas phase aeration method in the field

A trial production was carried out in Hokkaido (Kenbuchi-cho, Kamikawa-gun) in order to grasp the germination, growth and productivity of the tubers obtained in the present invention in the field. Tubers were prepared in the same manner as in Example 1. After storing for a certain period of time at low temperature (at 2 to 4) and in several places, tubers were grown according to local customary methods according to tuber size and storage period. However, the depth at which the tubers are planted was set slightly shallower than the conventional method. Only 0.1 to 0.5 g of tubers were germinated and raised in a greenhouse using a paper pot (diameter 5 cm, length 7.5 cm), and then planted in a field. The results are shown in Table 4.

(Margins below this page) Table 4 Storage period Size ¹ ) Number of individuals Germination rate ² ) Number of stems ³⁾ Yield ⁴⁾ High potato ⁵ 〉 High potato per 10a Starch value

(Mon) (%) Yield

(Strain / strain) (g / strain) (g / strain) Yield (t) Number Ζstrain (%) Small 30 16.7 1.1 970 728 3.4 7.7 10.7

9 Ψ on 1

0.ί 1. To A 1 Π 10 1 0

ο Large 32 32 12.5 1.3 1207 1046 4.8 8.2 13.6 ぺ -Paper 7 60 1.1 1108 932 4.3 10.9 13.3 Small 30 93.3 1.5 1324 1203 5.6 12.1 11.8

5 Medium 30 90.0 1.4 1106 1006 4.7 9.2 14.4

Large 32 93.8 1.2 1324 1150 5.3 10.7 13.4 ぺ -Pa-Pop 60 1.6 1207 1029 4.8 11.1 12.4 Small 30 100.0 1.5 1120 985 4.6 10.7 12.1

9 Medium 30 96.7 1.8 1286 1179 5.5 10.7 15.1

Large 32 93.8 2.7 1317 1249 5.8 11.2 14.2 Control ^β> 60 83.3 4.2 1211 1140 5.3 10.9 14.5

1) Paper-Po'not: 0.1-0.5g, Small: 0.5-lg, Medium: l_5g, Large: 5-10g,

Planting density of 72cmX30cni for each plot

2) 4 weeks after planting

3) Average value of 12 shares in each ward

4) Total yield of each plot Z

5) 40g or more

6) Normal seed potato (40-120g)

The sprouting rate was extremely low only in the two-month storage period, which is considered to be because the two-month period before sowing was insufficient to release dormancy. In the storage periods of 5 months and 9 months, the sprout rate was high irrespective of the size, and the initial growth after sprouting was generally inferior to the control, but recovered later. The final yield was close to the control in all the size groups, and in some cases was higher than that of the control. It was confirmed that the tubers produced by the present invention had high growth and production ability.

Example 4

Effect of carbon dioxide application in culture tank on subsequent tuber formation

The test materials used in this experiment were aseptic from growth point culture of bailecho (So lanum tuberosum L., cultivar: Vincci, obtained from the Agriculture, Forestry and Fisheries Gene Bank through the mediation business of the Organization for Promotion of Specified Industrial Technology Research Institute for Biological Systems). Plants were used. The same foliage growth medium 5 ^ as in Example 1 was placed in a culture tank having a diameter of 20 cm, a height of 24 cm and an internal volume of 8, and three plants each having 5 nodes and roots were placed thereon. Place the cultivation tank in an incubator capable of irradiating light from all sides, and cultivate first for 3 weeks under the conditions of 70001 UX illuminance, continuous illumination, 20 and air flow rate of 30 ral Z for liquid phase. Was performed. During this process, the plants grew well, mainly in the liquid, and formed numerous axillary buds. Some foliage extends to gas phase At this time, no stron was found. Then, while maintaining the same aeration conditions in the liquid phase, the gas phase was aerated with 10% carbon dioxide-enriched air at 100 ml / min and cultured for 3 weeks. By this cultivation, vigorous induction and proliferation of stomatones, mainly from the axillary buds in the liquid phase, were observed along with further growth of the plant. Many of the mouths grew obliquely toward the wall of the culture tank, but some grew slightly upward, and some reached the gas phase. Eventually, the plants in the liquid became a peculiar form covered with stron. As a control, a section was provided in which air not enriched in carbon dioxide was passed through the gas phase. No growth was observed in this control plot, such as obvious attraction of stron, and the difference in plant morphology from the carbon dioxide-treated plot was remarkable. Thereafter, the remaining medium was replaced with a tuber induction medium 1.5, and the cells were cultured at 20 ° C under continuous dark conditions for 4 weeks. In this case, ventilation was performed under the conditions of carbon dioxide-enriched air at 30 ml / min in the liquid phase and 1 ^ Z in the gas phase in both the CO2 application and control sections. In the carbon dioxide application zone, most of the stron formed in the liquid by this medium exchange was also exposed in the gas phase. The results are as shown in Table 5.

(Margins below this page) Table 5

W

Control ratio

In the CO2 application zone, from the second day after the medium was replaced, (1) most of the stron exposed to the gas phase started to tuberize at the tip almost synchronously regardless of the distance from the culture medium, Subsequently, (2) tubers were formed directly at the nodes of the strons or via newly derived strons. After that, (3) the occurrence of white stalks and strons and tubers similar to those in Example 1 were observed from the nodes of the other stems. Due to such tuber formation process, the plant in the carbon dioxide application section exhibited a form peculiar to the present invention in which tubers were densely present throughout the gas phase portion. On the other hand, tuber formation in the control plot occurred by the process of only the above (3), and its onset time was later than that in the carbon dioxide application plot. As a result, the total number of tubers per tuber and the weight of tubers were significantly higher in the group to which carbon dioxide was applied than in the control group. The average weight per tuber is smaller in the carbon dioxide-applied section, but it is clear from the reference examples that normal sprouting, growth and harvesting are possible from tubers of such a size.

Example 5

Effect of carbon dioxide application in the culture tank on subsequent tuber formation

By the same method as in Example 2, the foliage of the rasset burbank was grown for 3 weeks. Then, foliage growth medium whose sucrose concentration was changed to 2% was added 2 ^, and only aeration to the gas phase was performed using air enriched with 2% carbon dioxide. Consecutive days length, continued for a further 3 weeks of culture under the illuminance 60001 ux, 20 ^e C conditions, induction be sampled opening down, urged extension. The control group had the same ventilation conditions as in Example 2. After the addition of the culture medium, the light irradiation was performed in an incubator that allows light irradiation from the surroundings. Thereafter, the medium was replaced with a 1.5 ^ tuber induction medium, and tuber induction was performed at 20 for 4 weeks in the dark. In this case, the ventilation conditions were increased to two diagonally opposite vents for the gas phase in both the CO2 application and control sections, and 1 i /% from each, and 30 mlZ for the liquid phase. The results are shown in Table 6. Table 6

Contrast ratio As in the case of Binche in Example 4, a remarkable increase in the total number and total weight of the finally formed tubers was observed in the varieties ratchet bar bank as well, and the effect of carbon dioxide application during plant growth was reduced. It could be confirmed. Also, in the present results, the rate of increase in total weight relative to the control Because of this, the average tuber weight per piece also improved significantly. On the other hand, the quality of the obtained tubers was excellent because the tuber formation position was mainly in the gas phase in both the CO2 application and control plots, and both plots showed almost no hypertrophy and no damage to the epidermis. I was

Example 6

Effects of carbon dioxide application in a culture tank under low-temperature conditions during the long days and dark periods on the induction, growth, and subsequent tuber formation of stone mouth

Five sterile plants (each having 5 nodes and roots) of Vinche and Russet Burbank maintained under the conditions of illuminance of 6000 1 UX, 16 hours photoperiod, and 20 at 20 were used as test materials, and the sucrose concentration was 2 The plant was placed in an 8 ^ culture tank containing 3% of the foliage growth medium changed to%. These culture tanks were cultured in an incubator capable of irradiating light from below, mainly under the conditions of illuminance 6000 1 UX, 16 hours day length, light period temperature 22 and dark period temperature 10 and formed after 6 weeks. The number of strons was investigated. Ventilation was carried out at a rate of 100 ml / min and δΟπιΐ Ζmin, respectively, with air enriched with 2% carbon dioxide in the gas phase and normal air in the liquid phase. As a control, a section in which the same amount of normal air was ventilated into the gas phase (control section IT), and a section in which the temperature condition was 22 ° C in both the light and dark periods (control section I) were provided.

Next, with respect to vinch ヱ, increase of foliage in the same manner as above After propagation and stron propagation, the medium was replaced with tuber induction medium 1 ^. At 20 weeks the number of tubers formed under dark conditions was investigated after 4 weeks. Aeration conditions were as follows. In both the CO2 application zone and the control zone I, normal air was set to 1 minute in the gas phase and 30 mlZ in the liquid phase. When the medium was replaced, the vigorously proliferated to the liquid phase during the application of carbon dioxide, and many of the strons that were successfully exposed were exposed to the gas phase.

Table 7 shows the results of the survey, and Table 8 shows the number of tubers formed.

*… Number of formed stalks (numbers) In the CO2 application plots, both for Vinche and Lasset Burbank, after growing the stems and leaves for 2 weeks after the start of cultivation, the stront also began to generate stront, mainly from axillary buds in the liquid. On the other hand, in the control plot, no stront was generated in both plots I and I, and the effect of carbon dioxide gas application was extremely remarkable. The strons observed in this experiment were the same as those observed in Example 4. Compared to this, it was characterized by the fact that there were more strong skewed strons and the number of branched strons was larger.

Also, in experiments using plants that had already started to induce stron by carbon dioxide application, vigorous stron proliferation was observed immediately after implantation, and better results were obtained. Table 8

As in Example 5, the strons in the carbon dioxide applied plot almost immediately synchronized with the tuberization of the tip, regardless of the distance from the culture medium, immediately after exposure to the gas phase. Started. Secondary strons, which had already branched, also formed tubers for a short period of time. As a result, the difference in tuber formation from the control group was large.

Example 7

Effect of CO2 application time on stron induction, growth, and subsequent tuber formation

Approximately 1 cm long for each node , And placed on a 500 ml triangular flask containing 400 ml of foliage growth medium. The Erlenmeyer flasks were sealed using aluminum foil with a gas-permeable filter (trade name: PF Mikuguchi Filter-1: manufactured by Shibata Hario), and subjected to static culture under the following three conditions to induce stron induction and The extent of proliferation was investigated. (A) Cultivation for 6 weeks (control) at a temperature of 20, continuous lighting, an illuminance of about 7,0001 ux, and in the absence of carbon dioxide (atmospheric carbon dioxide concentration of about 0.03% (v / v)). (B) Culture for 6 weeks under the same conditions as (A), except that the culture is performed in an atmosphere with an increased carbon dioxide concentration of 2% (v / v). (C) After culturing under (A) condition for 3 weeks, further culturing under (B) condition for 3 weeks. Light irradiation was performed from all sides in each section. Thereafter, the medium remaining in the flasks was replaced with 100 ml of tuber induction medium, and the tuber was further cultured for 4 weeks under continuous dark conditions at 20, and the degree of tuber formation was examined and compared. The results are as shown in Table 9.

(Margins below this page)

Table 9

'Average value per container 4 replicates

Control ratio

As Table 9 shows, the effect was evident even under culture conditions in which the vessel was placed in an atmosphere enriched with carbon dioxide. In other words, in the control group (A), no stron proliferation was observed, whereas in the group that was under the conditions of carbon dioxide application from the start of culture ((B)), and in the middle of culture, carbon dioxide was applied. In the section ((C)) cultured under the conditions, vigorous stron induction and proliferation were observed. The tuber formation (B) and (C) were more than three times the number of tubers per container and almost twice the total weight of the tuber compared to the control. The tubers in section (A) started to form about 5 days after the medium was changed, and the number increased gradually over the next 3 weeks, whereas most of the tubers in section (B) and (C) It was formed by the strontium's tip swelling in synchrony over a short period of about 2 to 14 days, which also showed a significant difference from the control. Sections (B) and (C), that is, the difference between the application start time and application period of carbon dioxide gas, did not significantly affect the degree of stron growth and tuber formation in this experiment.

The average weight per tuber was lower in the CO2 application plots than in the control plots.However, as is evident from the reference example, the tubers had an extremely small size of about 0.1 lg. Since it has been confirmed that sprouting, normal growth, and reproduction of tubers are observed, application of carbon dioxide gas does not reduce the effect of improving tuber formation efficiency. Example 8

Effect of the amount of medium during tuber formation on tuber formation from straws grown under carbon dioxide gas application 200 ml of foliage in a cylindrical culture vessel of 6 cm in diameter, 15 cm in height and 380 ml in inner volume The growth medium was added and used for the next experiment. The same culture method as in Example 7- (B) was used for the cultivation under the conditions of bed material and carbon dioxide gas application. On the 5th week of culture, when the stron grew vigorously in the gas phase and liquid phase, ① 50 ml The medium was exchanged with a tuber induction medium of (2) 100 ml and (3) 200 ml, and the relationship between the amount of medium and the degree of tuber formation was investigated. The culture conditions after medium exchange are the same as in Example 7. The results are as shown in Table 10.

(Margins below this page)

Table 10

Average value per container

4 reps for each ward

Control ratio

Cultured under atmospheric conditions (CO2 concentration: 0.03%)

7 The effect of carbon dioxide application is greatly shown in the number of tubers formed per container as in Example 7. This result indicates that reducing the amount of medium during tuber formation compared to that during foliage growth further improves the number and weight of tuber formation. In addition, many of the tubers at that time extended into the liquid phase during stron growth and formed into strons exposed to the gas phase during tuber formation.

Example 9

Effect of carbon dioxide concentration on stron induction, proliferation, and subsequent tuber formation

Example 8 The title was investigated using the same method as in ①. The concentrations of the test carbon dioxide in the atmosphere are 1%, 5% and 10%. The results are as shown in Table 11.

(Margins below this page)

Table 11

CD

Average value per container

4 reps for each ward

Control ratio

Cultured under atmospheric conditions (carbon dioxide concentration 0.03%)

Regarding the number of containers per container where the effect of CO2 application was most remarkable, the control significantly exceeded the control in all the groups, and the 5% group showed the highest value, about 4 times that of the control. No significant difference between 1% and 10% sections o

Example 10

Influence of daily CO2 application time on subsequent tuber formation under continuous light irradiation

Example 7-The application time of carbon dioxide in (C) was set to (2 hours) 6 hours (3 hours) per day for 10 hours, and the remaining time was measured by culturing under the condition of no application of carbon dioxide and continuous lighting. Was investigated. The results are as shown in Table 12.

(Margins below this page)

Table 12

Average value per container

4 reps for each ward

Control ratio

Cultured under atmospheric conditions (carbon dioxide concentration 0.03%)

The effect of carbon dioxide application under continuous light irradiation was clearly observed from a short time of 2 hours per day, and it tended to increase as the application time increased. Example 1 1

Influence of daily CO2 application during the light period on subsequent tuber formation under low temperature conditions during short and dark periods

Example 7 Under the conditions of (C), the latter three weeks during foliage growth were shortened to 10 days (10 hours long) and low during the dark period (10 ° (: 20 during the light period), The test was performed for 2 hours, 6 hours or ③ 10 hours under the condition of applying carbon dioxide during the period.

(Margins below this page)

Table 13

t

* — Average value per container

4 reps for each ward

** Control ratio

*** Atmospheric conditions (carbon dioxide concentration 0.03%) and

Short day cultivation at low temperature

Under the short-day, low-temperature conditions used in this example, stron elongation was also observed in the control, but the number and thickness of the strontium were higher in the sections to which carbon dioxide was applied than in the control. Was. The effect of carbon dioxide on tuber formation was observed from a 2-hour treatment per day with a total weight per container and a 6-hour treatment per day with the number of tubers.

〔The invention's effect〕

ADVANTAGE OF THE INVENTION According to this invention, tuber with which storage property and cultivation property were improved compared with the conventional method can be mass-produced easily.

Claims

The scope of the claims

1. A method for producing a tuber of a Solanum plant having a tuber-forming ability, comprising a step including a foliage growing step and a tuber forming step in a culture vessel.

(1) In at least a part of the foliage growth step, a plant piece having at least one bud is converted into a liquid medium containing at least a carbon source and an inorganic salt, and at least part of a light period is carbon dioxide gas. Cultivated under the conditions applied to induce foliage and stron, proliferate,

(2) In the tuber forming step, at least a part of the stron formed in the liquid medium in the foliage growing step is exposed to a gaseous phase, and cultured in a liquid medium containing at least a carbon source. For producing tubers specializing in

2. The method for producing tubers according to claim 1, wherein in the tuber forming step, the relative humidity of the gas phase is reduced by aerating the gas phase to culture the tuber.

3. The method for producing tubers according to claim 1, wherein the applied concentration of carbon dioxide is 0.1 to 30% (v / v).

4. The method of applying carbon dioxide gas is characterized in that 0.1 to 30% (v / v) of carbon dioxide gas is aerated at a rate of 0.001 to 1 volume per minute with respect to the volume in the container. The method for producing tubers according to claim 1, wherein

A method for producing tubers of a Solanum genus plant having tuber-forming ability comprising a step including a foliage growing step and a tuber forming step in a culture vessel, wherein the tuber forming step is performed by aerating the gas phase in the tuber forming step. A method for producing tubers, which comprises culturing at a reduced relative humidity in the gas phase.